The present invention relates to viral vectors and viral DNA.
Adenovirus has been studied for its role in human disease (25), as a model for many important discoveries in molecular biology, including mRNA splicing, DNA replication, transcription and cell transformation (reviewed in 44) and more recently as a powerful reagent for transient gene expression (12, 46). A detailed understanding of the adenoviral life cycle is well established (reviewed in 50). Since the initial efforts to use adenovirus as a gene transfer vector (18, 52, 28) the virus has gained in popularity as a vector and a number of methods of generating alterations in the viral genome to carry novel genes have been developed (2, 5, 11, 15, 21, 23, 26, 32, 38, 41, 43, 48 reviewed in 31, 49). Because of the ease of vector construction and purification, and because these vectors have a potent ability to transiently transduce novel genetic material into a variety of mammalian cell types in vivo, adenovirus vectors were used extensively in early efforts at clinical gene therapy.
Unfortunately, several features of the adenovirus type 5 (Ad5) based vectors initially used have limited the success in the initial applications. These included both the host immune response to adenovirus (reviewed in ref. 55) as well as the failure of the virus to efficiently enter certain target cell types (20, 58, 59). Thus, there is now an interest in adenovirus types that could provoke less aggressive host immune responses and could enter target cells with greater efficiency.
A large number of alternate adenovirus serotypes are known and may provide advantages in some applications over Ad5-based vectors. Additional adenoviruses that have recently been modified as vectors include the ovine adenovirus 287 (29, 53, 56,), the bovine adenovirus type 3 (40, 60), and the canine adenovirus (30). It was considered that these alternate serotypes would provide both a novel vector backbone to which there is no pre-existing immune response in the target host. Furthermore, because adenoviruses are extremely species specific in their replication capacity (50) a degree of security against inappropriate vector replication is gained by using an vector derived from a distant species of adenovirus.
There are several justifications for pursuing these alternate viral subtypes. For vaccine applications in their non-human hosts, these viruses, if properly modified, may provoke more effective immune responses than a human adenovirus based vector. Furthermore, more robust immune responses might be expected from a replication competent virus; thus a vector is most useful in a host where replication is partially or fully permissive. This is not the case with human adenovirus based vectors in nearly all nonhuman hosts.
It has been an object of the invention to provide an alternative adenovirus vector for use as a gene delivery vector and for use as a vaccine.
To solve the problem underlying the present invention, the avian adenovirus CELO has been chosen to be modified. CELO (chicken embryo lethal orphan or fowl adenovirus type 1, reviewed in 39) was characterized as an infectious agent in 1957 (57). There are few serious health or economic consequences of CELO virus infection. CELO can be isolated from healthy chickens and in general, do not cause disease when experimentally re-introduced into chickens (10).
CELO virus is structurally similar to the mammalian adenoviruses (mastadenoviruses) with an icosahedral capsid of 70-80 nm made up of hexon and penton structures (33); the CELO virus genome is a linear, double-stranded DNA molecule with the DNA condensed within the virion by virus-encoded core proteins (33, 36). CELO virus has a larger genome than Ad5 (44 kb vs. ca. 36 kb, ref. 6, WO 97/40180). The CELO virion has two fibers of different lengths at each vertex (24, 33, 35) rather than the single fiber of most other serotypes (reviewed in 50). The CELO virus is not able to complement the E1A functions of Ad5 and CELO virus replication is not facilitated by Ad5 E1 activity (37). The complete DNA sequence of CELO (6, WO 97/40180) revealed additional differences between CELO virus and the mastadenoviruses including the absence of sequences corresponding to the Ad5 early regions E1A, E1B, E3 and E4. The CELO genome contains approximately 5 kb of sequence at the left end and 12 kb at the right end, rich in open reading frames, which have no sequence homology to Ad5 but probably encode the early functions of the virus.
When developing CELO into a gene delivery vector, it has been considered that the virus is naturally defective in mammalian cells and this property should limit the possibility of complementation by wildtype mammalian adenovirus. The CELO virion has increased DNA packaging capacity and much greater physical stability than the virion of Ad5. One practical feature of CELO is the ability to grow the virus in chicken embryos, a system of low cost and high convenience (9, 33).
For application in avian systems, especially for vaccine applications, it would be useful to have a CELO derivative with reduced replication capacity. Such a replication defective virus would allow transduction of avian species or avian cells similar to replication competent CELO vectors, but the amplification and spread of the modified virus would be limited by the impaired replication capacity of the virus.
A gene termed Gam1 was originally identified in the CELO genome in a search for viral genes that influence cell survival (7). The Gam1 protein is encoded by CELO nucleotides 37391-38239, transcriptional control sequences are located within ca. 1500 basepairs upstream and ca. 300 basepairs downstream of the coding region.
The present invention relates to recombinant CELO virus or CELO virus DNA that have the region spanning nucleotides 37391-39239 of the CELO wild type virus genome completely or partially deleted or altered or that contain an insertion in this region, any of which modifications results in a complete loss of Gam1 expression or prevents the expression of a functional Gam1 protein. Alternately to a disruption in the region defined above, the virus may contain a disruption in the transcriptional control elements of the Gam1 gene. (For simplicity, any modification that results in a complete loss of Gam1 expression or an inhibition of functional Gam1 expression will be referred to xe2x80x9cGam1 disruptionxe2x80x9d in the following.)
The suitability of a CELO mutation for obtaining a virus of the invention can be readily determined by ascertaining the absence of Gam1 expression. Suitable tests employ standard immunological methods, e.g. immunofluorescence microscopy or Western immunoblotting using antisera specific for the Gam1 protein.
The CELO nucleotide sequence numbering used in the present invention is derived from reference 6, WO 97/40180 and GenBank U46933, which describe the sequence of the wild type CELO virus genome.
CELO virus or CELO virus DNA with Gam1 disruptions, and their derivatives respectively, have been designated CELO AIM65 or CELO AIM65 derivatives, respectively.
In an embodiment, the invention is directed to a CELO AIM65 derivative with a complete or partial deletion and/or insertion within the Gam1 gene as defined above.
Preferably, the nucleotides 37391-39239 are completely deleted to provide more space for inserting the foreign DNA in place of the deletion.
In another preferred embodiment, the deletion comprises the region defined by the rescriction sites SmaI/Bgl II, i.e. the region spanning nt 36818-37972, which removes part of the Gam1 open reading frame.
The deletions defining CELO AIM65 may be combined with mutations defining CELO AIM46 (79) or its derivatives. Examples for AIM46 mutations are complete or partial deletion(s) of the regions spanning nt 41731-43684, 41523-43684, 41002-43684 or 40065-43684. Besides, the CELO AIM65 modifications may also be combined with the modifications described for AIM69 and/or AIM70 (79).
Thus, the CELO virus and CELO virus DNA of the invention carry a Gam1 disruption that is optionally combined with a deletion spanning approximately the region from nt 40,000 to approximately within 200 bp of the right terminus of the virus genome. The region that may be completely or partially deleted or disrupted is thus defined by the last three rightward open reading frames encoding peptides of greater than 99 amino acid residues, with the terminal repeat function of the virus normally residing within the last 100-200 bp of the virus genome remaining undisrupted. Optionally, the CELO virus and CELO virus DNA of the invention may, in addition, have a deletion in the region defined by the open reading for the CELO dUTPase (794-1330). Since CELO AIM65 and its derivatives defined above allow the insertion of large pieces of foreign DNA, they are useful as starting material for producing CELO virus vectors.
Apart from allowing the insertion of an expression cassette for genes, the recombinant CELO virus of the invention has been shown to be replication defective in cell culture.
It was a further object of the invention to provide a method for cultivation of the CELO AIM65 virus of the invention.
The disruption in the Gam1 gene renders CELO AIM65 extensively defective in replication. A study of the biological function of the Gam1 gene revealed that Gam1 expression leads to increases in the cellular levels of certain heat shock proteins. It was therefore hypothesized that an essential function of Gam1 is to upregulate a heat shock response during infection. Based on this hypothesis, it was tested whether heat shock applied to the infected cell could allow the replication of a CELO derivative lacking the Gam1 gene.
Surprisingly it was found that, although the Gam1 deletion severely impaired CELO virus replication, the functions of the Gam1 gene in virus replication could be provided by exposing the host cells to a heat shock. This novel type of complementation allows to grow viruses that otherwise are severely defective in their replication capacity.
The invention relates, in a further aspect, to a method for producing CELO AIM65.
The method comprises the following steps: Cells which support wildtype CELO replication (e.g. LMH cells, ATCC No. CRL-2117) are grown and exposed to a heat shock either before, simultaneously, or after infection with the CELO AIM65 (e.g. 10-1000 particles per cell). Under conditions suitable for cell cultivation and for a period of time sufficient for producing the desired number of viruses, e.g. after cultivation for 4 days at 37xc2x0 C., lysates of the cells are prepared and virus is prepared by standard methods. The heat shock treatment preferably comprises exposure to temperatures above 43xc2x0 C., preferably 45xc2x0 C., for period of time sufficient to complement the replication defect, preferably for 30-120 minutes, most preferably 90 minutes. The optimal conditions can be determined empirically in a routine series of virus growth experiments.
Furthermore, in vivo, in chicken embryos the recombinant CELO virus of the invention is highly defective for replication, i.e. the virus does not replicate when infected at levels below 107 particles per embryo, however when infected at higher multiplicities, wild type levels of the virus can be obtained. This property facilitates the growth of the defective virus for various applications.
Although replication deficient, the virus of the invention, when used at high multiplicities of infection in chicken embryos, grows to wildtype levels and forms, as the virus grown in cell culture under heat shock conditions, the basis of a replication-defective vaccine strain.
Since CELO AIM65 and its derivatives contain the same capsid components as CELO AIM46, CELO AIM65 and its derivatives possess the same ability as CELO AIM46 to introduce genes into a broad range of cell types including, in addition to avian cells, human, bovine, equine, monkey, murine, and canine cell types.
The cells that support CELO virus replication and are thus useful for CELO virus production, may be selected from immortalized cells like LMH (27) or from primary avian embryonic cells, in particular kidney or liver cells. To identify useful cell lines, cells are tested for infectability and the ability to amplify an inoculum of virus after heat shock of the host cell as described below.
Alternatively, once a sufficient stock of the defective virus is obtained from cell culture growth using the heat shock step, the recombinant CELO virus may be produced by introducing CELO virus into chicken embryos at sufficiently high multiplicities (i.e. greater than 107 particles per embryo).
An alternate method of growing CELO AIM65 or its derivatives is based on the observation that Gam1 functions can be partially replaced by overexpressing hsp40 or another gene upregulated by Gam1 in the host cell. Thus replication of AIM65 or derivatives can be obtained by coinfecting the host cells with a recombinant adenovirus directing the synthesis of hsp40 (e.g. Adhsp40) or by transfecting the host cell with a plasmid encoding hsp40. Alternately, the hsp40 expression can be directed by an hsp40 expression cassette inserted directly in the CELO AIM65 genome (e.g. CELOdGhsp40).
In order to produce recombinant a CELO virus genome with a Gam1 disruption, a plasmid bearing the genomic right end 13.3 kb fragment is modified to delete a portion of the Gam1 coding sequence and an expression cassette, e.g. a BamH1 CMV/luciferase/xcex2globin expression cassette is inserted by standard ligation cloning. This modifed region is built into a recombinant CELO genome by homologous recombination to produce a plasmid (designated pAIM65). This modified CELO genome is then released from the plasmid backbone by restriction digest and introduced by transfection into cells that support CELO virus replication. Useful levels of CELO AIM65 virus replication were found to occur only when using heat-shocked LMH cells.
Alternatively, recombination can be performed in avian cells that support AIM65 viral replication, e.g. heat shocked LMH cells, by introducing a modified CELO subfragment that contains a deletion/insertion with a second CELO fragment such that overlapping homology between the two fragments allows recombination to full length CELO genome bearing the desired deletion/insertion.
The replication defective vectors of the invention have vaccine applications in avian species where wildtype levels of CELO viral replication could produce unwanted toxicity or pathology. The ability to propagate AIM65 derived vectors in inexpensive chicken embryos when inoculated in sufficient quantities or by applying appropriate heat shock conditions (which can be determined by routine cultivation assays), facilitates production of large quantities of the vector for any of these applications.
For vaccine applications, the foreign DNA encodes one or more antigens eliciting an immune response in the individual. The antigen may be the natural protein derived from the pathogen, or an immunogenic fragment thereof, e.g. an immunogenic peptide.
To drive expression of the foreign DNA, an expression cassette can be used, which typically includes a promoter active in the target cells, the cDNA of interest, a polyadenylation signal and optionally an intron. Alternately, the DNA inserted into the modified CELO genome may include endogenous CELO promoters, introns and polyadenylation signals to drive expression of the cDNA of interest.
An example for a useful expression cassette, which can be prepared by conventional methods, is derived from a plasmid designated pPM7. It contains the Cytomegalovirus (CMV) immediate early enhancer/promoter followed by a short polylinker with PacI, HpaI and KpnI sites, followed by a rabbit xcex2-globin intron/polyadenylation signal. The CMV/xcex2-globin material may be derived from plasmids available in the art (e.g. from the plasmid pLuc (74), which carries the luciferase gene), modified by PCR to add flanking restriction sites, e.g. BamH1, and subsequently modified by homologous recombination to replace the luciferase cDNA with a PacI/HpaI/KpnI polylinker. The final BamH1 cassette can be cloned into pSP65 to generate pPM7. cDNAs to be cloned into CELO AIM65 derivatives are first cloned into pPM7 using the unique restriction sites (PacI/HpaI/KpnI). Subsequently a restriction or PCR fragment, e.g. a BamH1 fragment, is prepared containing the CMV promoter/cDNA/xcex2-globin unit which is introduced into PacI linearized pAIM65 by homologous recombination. The CMV and xcex2globin sequences provide homology for the recombination and the luciferase cDNA is thus replaced with the novel cDNA of interest.
The expression cassettes described above can be modified, e.g. by using, instead of the CMV enhancer/promoter, a variety of other viral or cellular promoters including, but not limited to the SV40 enhancer promoter, the Rous Sarcoma Virus long terminal repeat (RSV LTR), the human xcex2-actin promoter, the CELO virus major late promoter, the adenovirus major late promoter, the rat insulin promoter.
Alternatives to to the rabbit xcex2-globins intron/polyadenylation signal include, but are not limited to the intron/polyadenylation signals from SV40, introns and polyadenylation signals from other viruses and from cellular genes could also be used.
Alternately to using an expression cassette, the foreign cDNA may be a simple insert within a region defining CELO AIM 46 or the deoxyUTPase, thus using endogenous CELO regulatory sequences, e.g promotor, intron, polyadenylation signal.
In the case that two different foreign cDNAs are to be expressed from the CELO vector, e.g. cDNAs encoding two different antigens from a pathogen, the following strategies may be used: in a first embodiment, two gene expression cassettes (carrying different cDNAs and different regulatory sequences) can be inserted into the CELO genome. Alternately, an internal ribosome entry site (IRES) can be used to provide expression from two cDNAs using a single promoter, as described by e.g. 70; 67; 71; 72. Thus, a typical expression cassette for CELO AIM65 carrying two cDNAs to be expressed, includes a promoter, the first cDNA, an IRES, and the second cDNA followed by an optional intron and by a polyadenylation signal.
The foreign cDNA, e.g. antigen cDNA, can be isolated from the genomes of the pathogens by standard methods, e.g. by PCR or by restriction digest, optionally including reverse transcription to convert RNA to DNA, and introduced into a transfer vector carrying the regulatory sequences and unique restriction sites, e.g. the pPM7. Subsequently, this antigen expression unit a recombined into a linearized plasmid bearing the CELO genome and having the same regulatory sequences and corresponding restriction sites, e.g the plasmid pAIM65. The resulting CELO-AIM65 vector, carrying the antigen cDNA, can be grown and purified from chicken embryos.
Examples for antigens useful for vaccine applications are given in WO 97/40180, which is fully incorporated by reference herewith.
Further examples for antigens that may be carried by the virus for vaccination applications are antigens of the infectious bursal disease virus (IBDV; 64) and antigens of Chicken coccidia, e.g. Eimeria acervulina, Eimeria brunetti, Eimeria maxima, Eimeria mitis, Eimeria necatrix, Eimeria praecox and Eimeria tenella (61, 62, 63), examples for antigens are a parasite refractile body transhydrogenase, lactate dehydrogenase, Ea1A and EaSC2 (reviewed in 77).
Further examples for antigens are the glycoprotein C (gC, glycoprotein gIII) of the porcine pathogen pseudorabies virus (the causative agent of Aujeszky""s disease (75; 76; 69). A CELO AIM46 vector carrying gC can be used to elicit an anti-pseudorabies response in pigs.
A robust immune response is to be expected from a replication competent virus. However, under certain conditions, the replication of CELO derived vectors (e.g AIM46 vectors) may produce unsuitable levels of pathology in the host. Therefore, the replication defective vectors of the current invention may be useful at limiting the spread of the vaccine without compromising the initial entry and gene expression from the vector in the host. In this regard, the CELO vectors of the present invention, CELO AIM65 and its derivatives, are ideally suited for avian vaccine applications.
The recombinant CELO virus vectors of the invention are also useful in human vaccine or gene transfer applications. Wild type CELO is replication defective in mammalian cells, therefore the additional replication block generated by the removal of Gam1 expression provides further measure of security. Furthermore, the removal of Gam1 expression will lower the amount of background gene expression from the vector which may have unpredictable effects on the host cell or organism.
An additional argument for pursuing a non-human adenovirus comes from the experience with human adenovirus in human gene transfer applications. Pre-existing immune responses to human adenovirus can impair the initial transduction by human adenovirus based vectors or might exacerbate the cellular immune response to transduced cells. A patient may have no immune experience with an adenovirus from a distant species (although 2 of 7 patients had neutralizing antibodies to the canine adenovirus vector; 30) and initial transductions will not be compromised by the host response to viral antigens. Except for certain agricultural workers, a previous immune exposure to CELO antigens would not be expected in most of the human population. CELO vectors might therefore have an advantage over vectors based on more common human adenovirus serotypes.
An additional conceptual advantage of CELO based vectors of the invention is that CELO, like the bovine, ovine, and canine adenoviruses, is naturally replication defective in human cells. Thus, replication of these vectors will not occur in human patients even in the presence of a wildtype human adenovirus infection.
For gene therapy applications, the foreign DNA may comprise any one or more DNA molecules encoding a therapeutically active protein. Examples are immunomodulatory proteins like cytokines; receptors, enzymes, proteins effecting apoptosis, proteins modulating angiogenesis, e.g. sFLT, FGF receptors, etc. For tumor vaccine applications, the foreign DNA encodes one or more tumor antigens or fragments thereof, preferably in combination with a cytokine.
Examples for human vaccine applications, gene therapy and tumor vaccine applications are given in WO 97/40180, which is fully incorporated by reference herewith.
For vaccine applications, the vector of the invention may be packaged as an enteric coated dosage unit, or in an injectable form for intramuscular, intraperitoneal or subcutaneous injection. Alternately, the vector may be admistered as a paste or a fluid intranasally or intratracheally, as an aerosol or as an intraocular drop. The vector may also be supplied incorporated in feed pellets or in the drinking water.
The quantity of virus introduced per patient, animal or egg may range from 1 to 1012 particles.
The virus preparation may include a physiological buffered saline or HEPES buffered saline and may optionally be mixed with adjuvants such as vitamin-E acetate, oil/water emulsion, aluminium hydroxide, -phosphate or -oxide, mineral oil emulsions such as Bayol(R) or Marcol 52(R) and saponins.
Itmay be useful to use a freeze-dried form of the virus as a vaccine (78). The inclusion of a stabilizer such as 10% sucrose may be used with a controlled two-step drying process (78). Alternative stabilizers include carbohydrates such as sorbitol, mannitol, trehalose, starch, sucrose, dextran or glucose, proteins such as albumin or casein, or their degradation products.
In vaccine applications, in order to enhance the host immune response, the immune response elicited by the application of the CELO vaccine vector that carries a specific antigen, can be boosted by additionally adminstering the same antigen or an immunogenic fragment thereof. Preferably, the additionally administered antigen is recombinant; it can be obtained by standard methods or by the method described below that uses CELO vectors to obtain the recombinant proteins from eggs. The combined application of the vector and the antigen can be performed as described by (65, 73). Preferably, the recombinant antigen is administered, optionally together with an immunostimulating adjuvant, subsequent to the CELO vector.
In a further aspect of the invention, CELO virus is used for producing any protein of interest.
CELO AIM65 derivatives may have advantages over replication-competent CELO virus derivatives in that toxicity produced by the virus replication could limit protein production. Cells or embryos infected with AIM65 derivatives are expected to survive longer than cells or embryos infected with replication competent CELO derivative and thus, the production of recombinant proteins encoded by AIM65 derivatives is expected to be higher.
In this embodiment of the invention, the CELO virus, e.g. CELO AIM65 or its derivatives, is engineered, as described above, by introducing the cDNA or, preferably, an expression cassette, encoding the protein of interest into one of the insertion sites of the recombinant CELO DNA of the invention. Virus may be obtained by replication in suitable cells, as described above, and the recombinant virus is introduced, preferably by injection into the allantoic cavity of an avian embryo. Preferably, approximately 4xc3x97107 particles are introduced into the allantoic cavity of 7 to 9 day old chicken embryos, which are subsequently incubated for three to four days at 37xc2x0 C. The recombinant material is then recovered from the allantoic fluid, serum, yolk, amniotic fluid or from the embryo itself.
The protein of interest may be an intracellular or a secreted protein. In the case of a intracellular protein, the protein can be recovered by lysing infected cells that accumulate in the allantoic fluid. In the case of a secreted protein, the material can be recovered from various extracellular fluids of the embryo (allantoic fluid, amniotic fluid, serum, yolk) or, in analogy to the recovery of intracellular proteins, by lysing infected cells.
In a preferred embodiment, the protein of interest is expressed as a fusion protein comprising the protein and, as a stabilizing sequence, an immunoglobulin Fc domain. The secretion of the recombinant protein can be directed by the natural signal sequence from the protein, which may, in addition to the signalling function, have a stabilizing function. The Fc domain confers stability to the protein in the extracellular space and provides a protein sequence that can be used for affinity purification of the recombinant protein using, for example, Protein A or Protein A/G chromatography resins. Constructs that include an Fc domain for stabilization and are thus useful to be expressed from CELO, have been employed to make soluble forms of the FGF receptor 2 (sFGFr; 66) and the VEGF receptor 1 (sFLT; 68).
As alternatives to the signal sequences of FLT and the FGF receptor, signal sequences from or fusions with the proteins ovalbumin, conalbumin, avidin and lysozyme can be used. These are proteins that are synthesized in the liver and/or oviduct of chickens and accumulate within the egg. Thus, using part or all of the coding sequence of these proteins fused to a protein of interest are expected to yield secreted recombinant proteins that are stable within the developing embryo; furthermore, using sequences of this type, e.g. avidin, provides a tag that facilitates chromatographic purification.